Receptor signaling pathways are the subject of widespread research efforts. A better understanding of these signaling pathways will lead to the design of new and more effective drugs in the treatment of many diseases. Of particular interest are the growth factor and related receptor signaling pathways and their role in cell growth and differentiation. Binding of a particular growth factor to its receptor on the cell plasma membrane can stimulate a wide variety of biochemical responses, including changes in ion fluxes, activation of various kinases, alteration of cell shape, transcription of various genes and modulation of enzymatic activities in cellular metabolism.
In particular, upon binding an external ligand, a receptor may undergo auto-phosphorylation of specific tyrosine residues, and/or may phosphorylate other proteins. This tyrosine phosphorylation creates binding sites for cytoplasmic signaling proteins which have specific domains that recognize the phosphorylated tyrosine and adjacent residues. Once bound, these signaling proteins may in turn be activated. The activated signaling proteins then may effect downstream processes. Pawson and Gish, Cell 71:359-362 (1992).
SH2 (which stands for Src Homologous) domains are amino acid sequences that are similar to a 100-residue, non-catalytic region of the Src tyrosine kinase and are present in various signaling molecules. Sadowski et al., Mol. Cell. Biol. 6, 4396 (1986). SH2 domains are the functional protein motifs that bind tyrosine-phosphorylated targets by recognizing phosphotyrosine and specific adjacent residues. J. A. Escobedo et al., Mol. Cell. Biol. 11, 1125 (1991); L. C. Cantley et al. Cell 64, 281 (1991); T. Pawson and G. D. Gish Cell 71, 359 (1992); S. Zhou et al. Cell 72, 767 (1993); G. Waksman, S. E. Shoelson, N. Pant, D. Cowburn, J. Kuriyan Cell 72, 779 (1993). Activation of tyrosine kinases by growth factors, cytokines, and oncogenic agents therefore serves as a switch for assembling SH2 domain-containing proteins with their tyrosine-phosphorylated targets in signaling complexes, in which downstream effectors are activated.
The use of tyrosine kinase binding domains, including SH2 domains, has been discussed in methods for identifying targets of tyrosine kinases in cells, and thus identifying intermediates in cell signaling pathways. See, PCT Patent Application No. WO 92/13001, to Schlessinger et al.
The specific use of SH2 domains and subdomains in affecting the SH2 phosphorylated ligand regulatory scheme, or screening for compounds which affect SH2 binding in the SH2 phosphorylated regulatory scheme, as well as in assaying for the presence of SH2 binding phosphoproteins, has generally been described. See, U.S. Pat. No. 5,352,660 to A. J. Pawson.
Specific SH2 containing proteins include the products of the SHC gene. The SHC (which stands for SH2, Collagen) gene encodes a transforming protein expressed as 46-and 52-kD proteins, that are tyrosine phosphorylated in response to a number of growth factors, e.g., PDGF, EGF and FGF, and have been implicated as mediators of signaling from growth factor receptor and non-receptor tyrosine kinases to Ras. G. Pelicci et al. Cell 70, 93-104 (1992); M. Rozakis-Adcock et al. Nature, 360:689 (1992).
Thus, a great deal of attention has been directed toward studying these SH2 domains and their role in cell signaling pathways. However, SH2 domains, and the proteins which comprise them, are not the only phosphotyrosine binding mediators of such pathways. Accordingly, the study of these pathways, and the ability to control them requires identification and characterization of other phosphotyrosine binding domains. The present invention meets these and other needs.